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First published online October 27, 2006; 10.1104/pp.106.087783 Plant Physiology 143:108-121 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Evaluation of Source Leaf Responses to Water-Deficit Stresses in Cotton Using a Novel Stress Bioassay1,[OA]United States Department of Agriculture, Plant Stress and Germplasm Development Unit, Lubbock, Texas 79415
Water-deficit stresses preferentially reduce shoot growth, thereby disrupting the flow of carbohydrates from source leaves to the developing sinks. Here, we use a novel stress bioassay to dissect responses of field and greenhouse-grown cotton (Gossypium hirsutum) source leaves to water-deficit stresses. Fifth main stem leaf samples were harvested at sunrise and subjected to a prolonged elevated respiratory demand in the dark. Sucrose levels are lower in nonstressed cotton at sunrise compared to water-deficit stressed cotton, potentially predisposing the nonstressed tissue to succumb more rapidly. Tissue death was determined initially using the cell viability stain 2,3,5-triphenyltetrazolium chloride, but was determined in subsequent experiments by monitoring the decline in chlorophyll fluorescence yield. Fluorescence yield measurements were obtained within minutes of harvesting and individual samples were monitored over the time course of the treatment. Analyses of the time course and magnitude of chlorophyll fluorescence yield decline in samples from irrigated and dryland plots permitted the detection of stress responses within 24 h of the cessation of irrigation. The rate of fluorescence yield decline during the elevated respiratory demand treatment slowed as the water-deficit stress increased. Upon irrigation, the source leaves of the water-stressed plants recovered to prestress values within 4 d. Well-watered cotton overexpressing heat shock protein 101 had identical rates of fluorescence yield decline as nontransgenic cotton. These results suggest that the delayed decline in fluorescence yield of water-stressed tissue exposed to prolonged elevated respiratory demand can be used as a sensitive indicator of water-deficit stress responses.
Plants respond continuously to changes in available water, temperature, light, salinity, and other abiotic factors (Hasegawa et al., 2000  ; Turnbull et al., 2001; Chinnusamy et al., 2004; Zheng et al., 2004; Nixon et al., 2005). Physiological responses of plants generally vary with the severity and duration of the stress. The most sensitive processes are altered by a very mild stress and these changes intensify while additional processes become affected in accordance to their sensitivity to stress.
Historically, measurements of leaf or canopy temperatures have been used to evaluate the severity of water-deficit stress in plants (Jackson et al., 1988
Sensitivity to water stress depends upon the tissue in question. Under mild water stress, shoot growth is restricted while root growth continues (Sharp and Davies, 1979
Cotton (Gossypium hirsutum), like most C3 and C4 plants, accumulates photosynthate in the source leaves during the daylight hours and mobilizes these reserves at night to the growing points or sinks of the plant (Warner and Burke, 1993
This study evaluated the usefulness of a novel stress test bioassay in detecting the onset and magnitude of cotton water-stress responses under field and greenhouse conditions.
The study was conducted in Lubbock, Texas during the 2003, 2004, and 2005 growing seasons. The weather patterns during the 3 years of this study represented the driest year on record (2003), the wettest year on record (2004), and the average year (2005; Fig. 1 ). The 2003 growing season began with sufficient moisture to start the crop but failed to have any physiologically significant rainfall throughout the remainder of the season. The 2004 growing season had rain events throughout the season with four events of 15 mm or greater, and six events of approximately 10 mm. The 2005 growing season had rain events evenly spaced throughout the season with five events of 15 mm or greater.
Comparison of a Cell Viability Assay with the Stress Test Bioassay
A stress test was developed that easily allowed the evaluation of physiological responses of 200 to 300 field-grown plants per day per person. The stress test capitalized on the plant's inherent sensitivity to stress, the reduction in shoot growth associated with stress, and the impact of stress-induced growth reductions on source-sink relations. Initial studies collected leaf punches from source leaves at dawn, increased leaf disc respiratory demands by placing the tissue under elevated temperatures in the dark, and monitored the rate of tissue death using the 2,3,5-triphenyltetrazolium chloride (TTC) viability assay. TTC reduction has been used as a viability assay for over 50 years (Bennett, 1949
Impact of Sampling Time on the Stress Test Determination The initial premise for the stress test bioassay was that the large nighttime mobilization of photosynthate from source leaves to developing sinks of nonstressed plants would result in significantly less photosynthate remaining in the source leaves at dawn compared with the source leaves of stressed plants exhibiting reduced shoot growth. Figure 3 is a graph showing the time course of fluorescence yield decline at 39°C of irrigated (white symbols) and dryland (black symbols) cotton source leaf samples harvested at sunrise (7:30 AM) and sunset (7:30 PM) on day of year (DOY) 199 in 2004. Circles represent the samples harvested at sunrise and the squares represent the samples harvested at sunset. A significant difference in the rate of tissue decline between irrigated and dryland samples harvested at 7:30 AM was apparent within 30 min. The samples from the irrigated treatment declined from the initial CFY reading of 0.8 to a reading of 0.12 during the 6 h incubation. The samples from the dryland treatment, however, only declined from a CFY reading of 0.81 to a final reading of 0.33. Samples from the irrigated and dryland treatments harvested at 7:30 PM exhibited CFY values similar to one another during the first 2.5 h. The CFY of the sample from the irrigated treatment then declined rapidly between the 2.5 and 6 h readings resulting in a CFY reading similar to the CFY of the sample harvested from the irrigated treatment at 7:30 AM. The CFY of the samples from the dryland treatment harvested at 7:30 PM remained elevated compared with the sample from the irrigated treatment, ending with a CFY similar to the sample from the dryland treatment harvested at 7:30 AM. Because of these results, subsequent samples were harvested at dawn to enhance the rate at which treatment differences could be detected.
Carbohydrate Analyses
Leaf discs harvested in the morning from the fifth mainstem leaves of irrigated and dryland cotton were evaluated for Suc and starch contents to determine if differential levels of photosynthate could be detected between the treatments. Figure 4
shows the levels of Suc (black bars) and starch (white bars) in leaves of irrigated and dryland cotton. Suc levels increased and starch levels decreased in leaves of dryland cotton compared to the irrigated samples. Similar increases in Suc levels have been reported previously for water-deficit stress sensitive cotton lines (Timpa et al., 1986
Comparison of Varietal Water-Deficit Stress Responses in 2003 Six commercial cotton varieties were evaluated during the first year of this study. Figure 5 shows the graph of the time course of fluorescence yield decline of irrigated (white circle) and dryland (black circle) cotton source leaf samples on DOY 218 in 2003 harvested at sunrise and dark incubated at 39°C. Varietal differences were apparent using this bioassay in how the plants had responded to their environments prior to sampling. All of the varieties evaluated from the irrigated and dryland treatments exhibited an initial CFY value of 0.8 prior to the 39°C metabolic challenge and then exhibited a decline in CFY over the next 7 h of treatment. Suregrow 215 samples from the irrigated plants showed a gradual decline in CFY over the 7 h treatment with a final value of 0.256 being observed. The dryland sample showed a decline in CFY during the first 3 h of treatment, and then CFY values leveled off at a value of 0.575. PM2326 samples from the irrigated plants showed a gradual decline in CFY over the 7 h treatment with a final value of 0.314 being observed. The dryland sample showed a decline in CFY during the first 6 h of treatment, and then CFY values leveled off at a value of 0.501. FM989 samples from the irrigated plants declined in CFY over the 7 h treatment to a final value of 0.348. The dryland sample showed a decline in CFY during the first 4 h of treatment, and then CFY values leveled off at a value of 0.630. PM2280 samples from the irrigated plants declined in CFY over the 7 h treatment to a final value of 0.216. The dryland sample showed a decline in CFY to a value of 0.538. Beltwide 24 samples from the irrigated plants declined in CFY over the 7 h treatment to a final value of 0.275. The dryland sample showed a decline in CFY to a value of 0.568. Finally, DP555 samples from the irrigated plants declined in CFY over the 7 h treatment to a final value of 0.258. The dryland sample showed a decline in CFY to a value of 0.593.
The Dynamics of the Recovery from and Onset of Water-Deficit Stress Graphs of dryland Paymaster 2326 responses to watering and subsequent drying between DOY 219 and DOY 239 during the 2003 season are shown in Figure 6 . Initial readings of the irrigated (white circle) and dryland (black circle) cotton were taken on DOY 219 immediately before irrigating the dryland treatment for 4 d with 5 mm of irrigation per day via subsurface drip lines. The irrigation of the dryland plot was stopped on DOY 223 and the plot was allowed to dry during the next 2 weeks. The solid black line in the DOY 223, 224, 226, 231, and 239 graphs shows the response of the original dryland plants to watering and subsequent drying. The gray lines show the data obtained on DOY 219 for comparison. Within 4 d of irrigation, the previously water-stressed plants exhibited CFY declines identical to plants that had always received irrigation. One day following the cessation of irrigation the plants exhibited CFY declines that were slower than observed the day before. These results suggest that the plants sensed differential water availability and had responded to the developing water-deficit stress. The water-deficit stress intensified over the next 2 weeks with the plants receiving no additional irrigations and only 7 mm of rainfall.
The transition of the cotton plants from nonstressed to water-deficit stressed to nonstressed was observed in 2004 in response to seasonal rainfall events. Figure 7 shows the time course of fluorescence yield decline of irrigated (white circles) and dryland (black circles) Suregrow 215 cotton between DOY 190 and DOY 223. The field plots received 32 mm of rain between DOY 170 and 188 so that the dryland cotton evaluated on DOY 190 exhibited only a moderate level of stress compared with the irrigated plots. No precipitation over 1 mm occurred between DOY 190 and 204, and the graphs for DOY 194, 201, and 204 illustrate the development and maintenance of water-deficit stress over this time period. Rain events on DOY 205 and 207 provided 22 mm of water to the dryland plants. The DOY 208 graph shows a dramatic change in the CFY decline compared with DOY 204. Additional rainfall between DOY 209 and 222 provided an additional 48 mm of water to the dryland plants. Samples harvested on DOY 223 exhibited similar CFY declines between the irrigated and dryland plants.
A second study evaluated the transition from irrigated to water-deficit stressed cotton between DOY 236 and 244 of the 2004 growing season. Figure 8 shows the time course of fluorescence yield decline of irrigated (white circles) and dryland (black circles) Suregrow 215 during this evaluation period. Irrigation was turned off the morning of DOY 236 and the plots were allowed to develop a water-deficit stress over the next week. Samples taken from the previously irrigated plots on DOY 238 exhibited similar CFY declines to those of DOY 236. The rate of the CFY decline was somewhat slower initially, however, the final CFY value obtained after 6 h of treatment was the same as that seen for DOY 236. Samples from the previously irrigated plots taken on DOY 239 showed an even slower initial decline in CFY compared with either DOY 236 or 238, and the final CFY values were significantly above those values obtained on DOY 236 and 238. The differences in CFY decline between the irrigated and dryland plots became smaller as the plants from the previously irrigated plot became more severely water-deficit stressed. No differences in the CFY decline were observed between the previously irrigated and dryland plots on DOY 244.
Spatial Variability Effects on Subsurface Drip Irrigated Cotton The sensitivity of the new stress bioassay provides a useful tool in evaluating the impact of spatial variability within an irrigation treatment. Figure 9 shows time course of fluorescence yield decline of irrigated (white circles) and dryland (black circles) Suregrow 215 cotton between DOY 236 and DOY 238 of the 2004 season. The photograph shows the location of samples harvested from the irrigated plot on DOY 236 (top arrow), DOY 237 (bottom arrow), and DOY 238 (both arrows). Dryland samples harvested from a single location on DOY 236, 237, and 238 showed similar CFY declines across all dates. The samples from the irrigated plots, however, showed CFY decline characteristics of a well-watered crop on DOY 236, and a more stressed appearance in the DOY 237 samples. The samples were harvested from a single row and the sampling locations were approximately 5 m apart. The samples harvested on DOY 238 from the same location within the irrigated plot as the samples shown in the graph for DOY 236 showed CFY declines similar to the DOY 236 samples. The samples harvested on DOY 238 from the same location within the irrigated plot as the samples shown in the graph for DOY 237 showed CFY declines identical to the DOY 237 samples. These results show the high degree of reproducibility of the bioassay and illustrate the need for caution when analyzing field-grown materials within a single irrigation treatment.
Comparison of Varietal Water-Deficit Stress Responses in 2005 In light of the findings that spatial variability can have significant impact on the outcome of the stress bioassay, the evaluation of genetic diversity in the stress responses of commercial cotton lines was reevaluated. Samples were harvested randomly along the treatment rows, and plots were alternated within the field. Figure 10 shows the time course of fluorescence yield decline of irrigated (white circles) and dryland (black circles) FM989, SG215, DP444, and FM800 cotton sampled on DOY 237 of the 2005 season. Clear varietal differences in plant status are observed in both the irrigated and dryland treatments. The three broadleaf cotton varieties from the dryland treatment (FM989, SG215, and DP444) had higher CFY values after the 5.5 h heat treatment than that of the okra leaf variety (FM800). The CFY decline in the DP444 irrigated samples was greater than the FM989 and SG215 irrigated samples. The okra leaf variety CFY decline in the irrigated treatment was less than any of the broad leaf samples.
Evaluation of Water-Deficit Stress Responses in Greenhouse-Grown Cotton The responses of cotton to imposition of and release from water-deficit stress in the field revealed physiological responses occurring over the time course of days to weeks. A series of experiments were performed to determine if similar temporal changes in stress responses occurred in greenhouse-grown cotton grown with more restricted rooting volumes than that of the field. Figure 11 shows that greenhouse-grown cotton grown under well-watered and limited-water conditions exhibit CFY declines similar to those of irrigated and water-deficit stressed field-grown plants. To evaluate the time course of the plant's response to a sudden severe water-deficit stress and recovery from the stress, cotton plants were grown to the 8 to 10 main stem leaf stage in 16-cm diameter pots. Samples were harvested from the well-watered plants and the CFY declines were determined (Fig. 12A ). Immediately after the samples were harvested the watering system was turned off and the plants were allowed to undergo a rapid and severe water-deficit stress for 24 h. All fully expanded leaves on the plants exhibited severe wilting after the 24 h water-deficit stress. Samples were harvested and the CFY decline analyzed (Fig. 12B). The plants were rewatered immediately after harvesting the samples and the response of the cotton plants monitored for an additional 7 d.
The pattern of CFY decline from five replicate plants showed the rapid decline in the well-watered plants similar to the irrigated field-grown plants (Fig. 12A). The severely wilted leaves sampled after a 24 h water-deficit stress showed only a slight change in the rate of the CFY decline compared with the well-watered plants (Fig. 12B). The characteristic response to water stress exhibited by a slow CFY decline in the bioassay was not apparent until the next morning (Fig. 12C), 24 h after the plants had been rewatered. A CFY decline identical to the 24 h recovery samples was observed after 48 h of recovery from the water stress (data not shown). It was only after 72 h post watering that the rate of CFY decline increased (Fig. 12D), suggesting that physiological recovery was under way. The plant's further recovery from the stress was suggested by the enhanced rate of CFY decline 96 (Fig. 12E) and 120 h (Fig. 12F) after rewatering.
Enhanced heat tolerance has been attributed to the constitutive expression of heat shock protein 101 (hsp101) in Arabidopsis (Arabidopsis thaliana; Queitsch et al., 2000
Throughout the day as much as one-half of the carbon assimilated by photosynthesis is stored in the chloroplast as starch. At night, this transitory starch is degraded, the degradation products exported to the cytosol, converted to Suc, and exported from the leaf (Geiger and Batey, 1967 ). In nonstressed plants only a small amount of starch is left at the end of the night (Warner and Burke, 1993). Under mild water stress, shoot growth is restricted while root growth continues (Sharp and Davies, 1979; Klein et al., 1986). We have capitalized on these characteristic plant responses to water-deficit stress in the development of a novel bioassay for evaluating plant stress. Leaf punches from source leaves of well-watered and water-deficit stressed plants were placed on a metabolic treadmill by prolonged exposure to elevated temperatures in the dark. Increases in temperature are known to increase plant tissue respiration, as exemplified by a recent study of soybean (Glycine max) leaves that showed that respiration increased by a factor of 2.5 between 18°C and 26°C average night temperatures (Bunce, 2005). The research described in this study evaluated the ability of source leaf tissues harvested at sunrise from well-watered and water-deficit stressed cotton to withstand prolonged respiratory demands before experiencing complete tissue death.
TTC reduction has been used as a viability assay for over 50 years (Bennett, 1949 CFY measurements used in this study were not used to show water stress effects on photosynthetic capacity. Despite difference in water-stress levels, all samples analyzed had acclimated to their environment to provide optimum photosynthetic capacity at dawn. This is exemplified by the 0.8 CFY value obtained for all samples regardless of water stress level analyzed over the 3 years of the study (see Figs. 3 and 5–13). Rather, the fluorescence measurements of the leaf samples before and during exposure to elevated respiratory demands in the dark were used as a surrogate for cell viability. The relationship between cell viability determined using TTC and the CFY (Fig. 2) justified our use of this tool to provide a rapid, nondestructive, measure of the viability of the tissue.
Throughout the day carbon assimilated by photosynthesis is stored in the chloroplast as starch and at night, this transitory starch is degraded, the degradation products exported to the cytosol, converted to Suc, and exported from the leaf (Geiger and Batey, 1967
Clearly, factors other than carbohydrate reserves (Fig. 4) also contribute to the overall viability of the tissues. Changes in membrane composition (Wilson et al., 1986
Varietal differences among cotton responses to water availability and water deficits in the field have been reported previously (Quisenberry and McMichael, 1991
The results of this study also identified varietal differences in cotton responses to available soil water. Differences in the rate of CFY decline among six commercial cotton varieties are shown in Figure 5. The observed differences occurred in both the irrigated and water-deficit stressed treatments. The significance of the observed differences was brought into question, however, after we identified the significant impact of field spatial variability on the overall well being of the cotton within a single variety. The spatial variability of crop water status results from variability of soil, crop canopy, topography, irrigation level (either inherent in the application method or caused by malfunctions), or from other factors such as salinity, which typically is spatially variable (Cohen et al., 2005
The impact of leaf shape has been evaluated previously for CO2 exchange rates of whole plants of four cultivars (FiberMax 832, Stoneville 474, DeltaPine 5690, and Paymaster 1220) measured at eight different temperatures (6°C–34°C) in 4°C steps (Bednarz and Van Iersel, 2001
In this study the FM800 showed a greater level of stress for the irrigated treatment exemplified by the CFY decline to a value of 0.3 while the broad leaf varieties showed declines between 0.1 and 0.2. It is possible that the daily exposure to air temperatures between 30°C and 36°C may have had a greater impact on the FM800 than the broad leaf varieties similar to the findings of Bednarz and Van Iersel (2001)
This study evaluated the time course of physiological responses to water-deficit stress in the field using the newly developed stress bioassay. The time course graphs in Figures 6, 7, and 8 show the progression of the onset of field-grown cotton water-deficit stresses and the recovery from these stresses in response to either irrigation or rain events.
The source leaves of greenhouse-grown cotton also showed the changes in the rate of the CFY decline in response to reduced water availability (Fig. 11). The response of the plants grown in a restricted soil volume to a sudden, severe water stress showed only a slight change in the rate of fluorescence yield decline during the stress process and suggests a continued mobilization from the source to the sinks (Fig. 12, A–B). The slow rate of fluorescence yield decline, characteristic of a water-deficit stress, was not observed until 24 h after the pots had been rewatered (Fig. 12C). It is interesting to speculate that the delay in the stress phenotype, and recovery from the stress may relate to the time required for the stress-induced signals to be synthesized and transported in sufficient concentrations to reduce sink growth (Sharp and LeNoble, 2002
The stress bioassay uses the enhanced respiratory demand associated with an elevation in ambient temperature as the engine behind this stress treadmill. A question that arises from the technique is what role do hsps play in the observed slowing in the rate of CFY decline associated with water-deficit stress. Association of hsp accumulation with water-stressed field-grown cotton was first observed by Burke et al. (1985b)
Crop Management: 2003 Eight 61-m rows of Suregrow 215 RR/BG, Fibermax 989 BR, DP 555 BG/RR, Beltwide-24R, PM 2326 BG/RR, and PM 2280 BG/RR were planted in a North-South orientation on DOY 149 (May 29, 2003) using a John Deere 7300 MaxEmerge 2 VacuMeter Planter. The plants received 5 mm of water per day per acre from underground drip lines located on 1-m centers. Roundup (Monsanto) was sprayed over the top according to manufacturer's instructions on DOY 168 for additional weed control. Pix (BASF Corporation) was sprayed over the irrigated crop according to manufacturer's instructions on DOY 217. The plots were sprayed with Ginstar (Bayer CropScience, RTP) according to manufacturer's instructions on DOY 273, and the plots were harvested on DOY 304.
Sixteen 61-m rows of Suregrow 215 RR/BG were planted in a North-South orientation on DOY 194 (May 12, 2004) using a John Deere 7300 MaxEmerge 2 VacuMeter Planter. The plants received 5 mm of water per day per acre from underground drip lines located on 1-m centers. Roundup was sprayed over the top on DOY 145 for additional weed control. Pix was sprayed over the irrigated crop on DOY 203. The plots were sprayed with Ginstar on DOY 289, and the plots were harvested on DOY 320.
Four 61-m rows of FM800, DP444, Suregrow 215 RR/BG, and FM989 were planted in a North-South orientation per replication on DOY 158 (June 7, 2005) using a John Deere 7300 MaxEmerge 2 VacuMeter Planter. Six replications were plants with three replications only receiving rain after planting and three replications fully irrigated. The plants received 5 mm of water per day per acre from underground drip lines located on 1-m centers. Pix was sprayed over the irrigated crop on DOY 206. The plots were sprayed with Ginstar on DOY 280, and the plots were harvested on DOY 306.
The U.S. Department of Agriculture-Plant Stress and Water Conservation Meteorological Tower is located immediately adjacent to the experimental plots. Five-minute measurements of wind speed (m/s), rainfall (mm), and temperature (C) were collected and hourly averages calculated.
Suregrow 215 RR/BG seeds were planted into 16-cm diameter pots containing 900 g of Sunshine Mix #1 soil (Sun Gro Horticulture Distributors). Three seeds were planted per pot and five pots were placed on benches in a greenhouse set to provide a 31°C/27°C day/night cycle. Plants were grown throughout the year and 430 W high-pressure sodium lights (P.L. Light Systems) were used to maintain a 16/8 h photoperiod. Nutrients were maintained by daily application with Peters Excel fertilizer (Scotts-Sierra Horticultural Products Company) through the automated watering system. Water-stress experiments on 2- to 3-week-old seedlings were performed by first thinning seedlings 1 week following planting to one plant per pot. Pots were saturated with water, excess water was allowed to drain from the pot overnight, and the pots were sealed from both ends with Uline 2 Mil poly bags (S-3478; permeable to air but impermeable to water). A small hole was cut in the top bag through which the cotton (Gossypium hirsutum) plant was allowed to grow. The hole was further sealed with Scotch packing tape and covered with a layer of dry potting mix to reduce heat load within the pot. The poly bags were fixed on the pots with a rubber band and strengthened with Scotch packing tape. The plants were allowed to grow for 18 d, at which time severe leaf wilting was observed at midday. Following analysis of the plant response to the water-deficit stress, the plants were allowed to recover from the stress by removing the poly bags, saturating the soil with water, and placing the pots on the automated watering system. Water stress and recovery experiments on 5- to 6-week-old seedlings were performed by turning off the automatic watering system for 24 h on the plants described above. The watering system was turned on following the 24 h stress treatment, and remained on for the remainder of the experiment.
Leaf punches were excised from the fifth main stem leaves when the plants reached the eight-leaf stage. Five replicate punches obtained from well-watered and water-deficit stressed plants were evaluated following incubation at 39°C using an OS1-FL Pulse Modulated Fluorometer (Opti-Science Corporation). Following analysis of the PSII CFY (Fv/Fm'), leaf punches immediately were transferred to 6 mL of 0.1 M phosphate buffer, pH 7.0, containing 0.8% TTC and incubated in the dark at 32°C for 24 h. Following the incubation, the TTC was extracted from the leaf punches with 95% ethanol and TTC reduction determined according to the procedure described by Burke (1998)
Leaves were taken from the youngest fully expanded leaf (fifth mainstem leaf from the top) of irrigated and dryland grown cotton in 2003. Immediately upon removal, the leaves were wrapped in aluminum foil and frozen in liquid N2 within 10 s. The frozen tissue was stored in a freezer at –80°C. Leaf discs were taken from the youngest fully expanded leaf (fifth mainstem leaf from the top) of greenhouse-grown cotton that had been well watered and cotton that had water withheld for 24 h. Samples from well-watered and water-stressed greenhouse-grown cotton were analyzed following 0, 3, and 6 h of dark incubation at 39°C. Starch was assayed according to Rufty and Huber (1982)
At sunrise, a leaf punch was harvested from a source leaf (in cotton this is the fifth main stem leaf from the top) using a number 6 cork borer and rubber stopper. This was repeated on five separate plants. The punches were transferred to a well in a Costar 3524 24-well cell culture cluster (Corning) that had been half filled with water. The lid was returned to the cell culture plate immediately following additional of the leaf punches. This process was repeated until samples from all treatments had been harvested. Upon returning to the lab, the punches were placed on moistened model 583 Gel Dryer Filter Paper (Bio-Rad Laboratories) in a Pyrex baking dish. The leaf punches and filter paper were covered with Glad ClingWrap (CO2 permeable; Glad Products Company) and pressed flat with a speedball roller for Microseal film (MJ Research) to remove air bubbles and ensure good contact between the tissue and filter paper. Initial Fv/Fm' levels were determined using an Opti-Science OS1-FL Modulated Fluorometer and then samples were placed in the dark in a VWR model 2005 incubator (Sheldon Manufacturing) set to 39°C. The samples were evaluated every 30 min after being placed in the 39°C incubator. The decline in Fv/Fm' over time was used as a relative measure of the stress level of the plant (a slow decline occurring in tissue from stressed plants, and a more rapid decline occurring in tissue from less stressed plants).
A gene reported to play a crucial role in thermotolerance (Queitsch et al., 2000
The binary vector pE1801-ocs/mas superpromoter-HSP101 was introduced into the EHA 105 strain of Agrobacterium tumefacians by direct transformation as described by Walkerpeach and Velten (1994)
Leaf samples from the putative transformants were evaluated for the presence of the superpromoter-HSP101 coding region. PCR analyses were performed according to the procedure of Xin et al. (2003)
Cotton leaves were ground into fine powders in liquid nitrogen and then extracted in 400 µL 2x SDS sample buffer (Laemmli, 1970
The author thanks Jacob Sanchez, Dee Dee Laumbach, Kay McCrary, and Julia Veyro for their excellent technical assistance. The author also thanks James Golden and John Stout for collection of the meteorological data. Received August 1, 2006; accepted October 22, 2006; published October 27, 2006.
1 Mention of a trademark, warranty, proprietary product, or vendor does not constitute a guarantee by the U. S. Department of Agriculture and does not imply approval or recommendation of the product to the exclusion of others that may be suitable.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: John J. Burke (jburke{at}lbk.ars.usda.gov).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.106.087783 * E-mail jburke{at}lbk.ars.usda.gov; fax 806–723–5272.
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-naphthaleneacetic acid + 0.1 mg/L kinetin +266 mg/L cefotaxime]. Hypocotyls were transferred to fresh MS2NK CL medium 3 weeks following Agrobacterium infection. Four weeks after the transfer, calli were cut from the hypocotyl ends and moved onto MS2NK 1/4CL medium [4.4 g/L Murashige and Skoog medium with Gamborg's vitamins + 2 g/L phytagel + 30 g/L D-(+)-Glc + 2 mg/L